ABSTRACT
Pseudomonas aeruginosa
undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO
3
−
) as an alternative electron acceptor. Understanding the ...mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against
P. aeruginosa
biofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B
12
to be a molecule that can reinstate defective anaerobic growth of
P. aeruginosa
. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B
12
, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B
12
, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of
P. aeruginosa
with normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B
12
, further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth of
P. aeruginosa
and illustrates the ability of vitamin B
12
to modulate the biofilm-forming capacity of
P. aeruginosa
under such condition.
Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key process for ...the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.
Inflammasome signaling can contribute to host innate immune defense against bacterial pathogens such as
. However, bacterial evasion of host inflammasome activation is still poorly elucidated. Quorum ...sensing (QS) is a bacterial communication mechanism that promotes coordinated adaptation by triggering expression of a wide range of genes. QS is thought to strongly contribute to the virulence of
, but the molecular impact of bacterial QS on host inflammasome defense is completely unknown. Here, we present evidence that QS-related factors of the bacterial secretant (BS) from
can dampen host inflammasome signaling in mouse bone marrow-derived macrophages. We found that BS from QS-defective Δ
mutant, but not from wild-type (WT)
, induces robust activation of the NLRC4 inflammasome.
-released flagellin mediates this inflammasome activation by Δ
secretant, but QS-regulated bacterial proteases in the WT BS impair extracellular flagellin to attenuate NLRC4 inflammasome activation.
-secreted proteases also degrade inflammasome components in the extracellular space to inhibit the propagation of inflammasome-mediated responses. Furthermore, QS-regulated virulence factor pyocyanin and QS autoinducer 3-oxo-C12-homoserine lactone directly suppressed NLRC4- and even NLRP3-mediated inflammasome assembly and activation. Taken together, our data indicate that QS system of
facilitates bacteria to evade host inflammasome-dependent sensing machinery.
We suggest an electrochemiluminescence (ECL)-sensing platform driven by ecofriendly, disposable, and miniaturized reverse electrodialysis (RED) patches as an electric power source. The flexible RED ...patches composed of ion-exchange membranes (IEMs) can produce voltage required for ECL sensing by simply choosing the appropriate number of IEMs and the ratio of salt concentrations. We integrate the RED patch with a bipolar electrode on the microfluidic chip to demonstrate the proof-of-concept, i.e., glucose detection in the range of 0.5–10 mM by observing ECL emissions with naked eyes. The miniaturized RED-powered biosensing system is widely applicable for electrochemical-sensing platforms. This is expected to be a solution for practical availability of battery-free electrochemical sensors for disease diagnosis in developing countries.
The inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on virulence phenotypes and gene expression regulated by quorum sensing (QS) in Escherichia coli O157:H7 were ...demonstrated at concentrations of 1 to 100 μg/ml, which are lower than the MIC (539 ± 22 μg/ml). At 25 μg/ml, the growth rate was not affected, but autoinducer 2 concentration, biofilm formation, and swarm motility decreased to 13.2, 11.8, and 50%, respectively. Survival at 5 days of nematodes (Caenorhabditis elegans) that were fed the pathogen without and with EGCG were 47.1 and 76%, respectively. Real-time PCR data indicated decreased transcriptional level in many quorum sensing-regulated virulence genes at 25 μg/ml. Our results suggest that EGCG at concentrations below its MIC has significant antipathogenic effects against E. coli O157:H7.
A novel pump-free miniaturized reverse electrodialysis (RED) system was designed to provide lasting power transduced from salinity gradients, named solid salt RED (ssRED), and this quasi-battery uses ...a solid salt instead of electrolyte solution for streamlined usage. It is portable, flexible, comparable in size to a universal serial bus flash drive, and easily activated with a small amount of water. It maintains a constant ionic concentration gradient through precipitation reactions between a pair of different salts. This precipitation-assisted solid salt RED (PssRED) is an unprecedented ionic power source as it can generate steady electricity in the absence of a driving pump. The PssRED was successfully coupled with bipolar electrode (BPE) microchip sensors which require stable ionic electricity and a polyelectrolyte ionic diode to realize a fully ionic circuit. It is envisioned that the range of application could be expanded to supply electromotive force to various devices through an ionic charge flow.
•C. elegans fed on EHEC O157:H7 wild-type or mutant strains.•Mutants lacking either pO157 or LPS O-side chains caused full attenuation in killing C. elegans.•The LPS O-side chain-defective mutant ...strain could not colonize in the intestine of C. elegans.•pO157 and PerA are required for EHEC O157:H7 to kill C. elegans.
As a model host, the nematode Caenorhabditis elegans has been used for studying unknown pathogen–host interactions and identifying novel virulence factors in bacterial pathogens. Among the bacterial pathogens that can induce death of C. elegans is enterohemorrhagic Escherichia coli (EHEC) O157:H7, a major serotype of EHEC that causes hemorrhagic colitis and hemolytic uremic syndrome in humans and animals. However, it is unknown which EHEC O157:H7 factors are required for nematode death. In this study, bacterial ability to kill C. elegans was tested for several EHEC O157:H7 wild-type and mutant strains missing one virulence-associated factor, including Shiga toxins, enterohemolysin, pO157 (a large virulence plasmid in EHEC O157:H7), Type 3 secretion system, LuxS, and lipopolysaccharide (LPS) O-side chains. Our results demonstrate that only mutants lacking either pO157 or LPS O-side chains cause full attenuation in killing C. elegans. The LPS O-side chain-defective ΔperA mutant strain was not able to colonize in the intestine even at 24h post-feeding with C. elegans, while the wild-type strain began to accumulate and colonize in the intestine as early as 3h post-feeding. A simple complementation of the mutant strain with the plasmid carrying the intact perA gene in trans completely restored the production of LPS O-side chains, as well as the ability to kill C. elegans. Our results show that pO157 and PerA are required for EHEC O157:H7 to kill C. elegans.
Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic ...importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgC and speC genes showing multiple mutations in the sequenced S. enterica subsp. enterca serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n=57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates.
Fifteen nonrepetitive ampicillin-resistant Salmonella spp. were identified among 91 Salmonella sp. isolates during nationwide surveillance of Salmonella in waste from 131 chicken farms during 2006 ...and 2007. Additional phenotyping and genetic characterization of these 15 isolates by using indicator cephalosporins demonstrated that resistance to ampicillin and reduced susceptibility to cefoxitin in three isolates was caused by TEM-1 and DHA-1 β-lactamases. Plasmid profiling and Southern blot analysis of these three DHA-1-positive Salmonella serovar Indiana isolates and previously reported unrelated clinical isolates of DHA-1-positive Salmonella serovar Montevideo, Klebsiella pneumoniae, and Escherichia coli from humans and swine indicated the involvement of the large-size plasmid. Restriction enzyme digestion of the plasmids from the transconjugants showed variable restriction patterns except for the two Salmonella serovar Indiana isolates identified in this study. To the best of our knowledge, this is the first report of the presence of the DHA-1 gene among Salmonella spp. of animal origin.
Herein, we introduce a simple route to fabricating hydrophilic microfluidic chips with an alternative material, a UV‐cured polyurethane‐related polymer, known as Norland Optical Adhesive (NOA 63). ...Conventionally, polydimethylsiloxane (PDMS) is widely used to fabricate microfluidic chips as an alternative to glass or SiO2 because PDMS is easily molded and relatively cheap. However, despite these advantages, the hydrophobicity of PDMS entails critical problems when it is used in microfluidic chips because microchannels inside the microfluidic chips, which have extremely low surface tension, are difficult to fill with aqueous solution without an extra pumping system. To overcome these problems, significant efforts have been focused on developing procedures to change the PDMS surface to be hydrophilic. However, the resulting hydrophilicity is generally short‐lived and the modification procedures require cumbersome multi‐steps. In the present study, we demonstrate that microchannel‐molding and microfluidic chip construction are easier using NOA 63 than when using PDMS and that the hydrophilicity of the NOA surface, which is induced by treatment with O2 plasma, lasts longer, for at least one month. Due to the longer lasting hydrophilicity, microchannels in NOA 63 microfluidic chips are spontaneously filled with solution by capillary reaction without any extra pumping over the period. The feasibility of NOA 63‐based microfabrication is verified by demonstrating NOA 63 microfluidic platforms with antibody‐immobilized beads for immunoassays.
Capillary forces can be used to fill this commercially available UV‐cured photopolymer with aqueous solutions (see figure) as opposed to the more commonly used polydimethylsiloxane. The beauty of this is that microfluidic chips created from this material can easily be produced by a simple oxygen‐plasma treatment. The chips in their turn can then be used for assays and other optical detection systems.
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